The invention relates to methods for the rapid industrially advantageous analysis of flavonoid and steroidal glycosidic compounds in natural products. Existing methods of equivalent analytical power are cumbersome, time consuming and expensive as well as being difficult to implement in the context of the contract research laboratory. The analysis of flavonoid and steroidal glycosidic compounds in HOSTA leaves served as model system.
Since ancient times a vast number of natural remedies of plant and animal origin has been used for medical treatment and disease prevention (Shibata, S., The Chemistry of Chinese Drugs, American Journal of Chinese Medicine (1979) 7(2): 103-141). The earliest known recorded recommendation of plants use to fight cancer appeared in the Ebers papyrus of Egypt dating from 1550 BC but this document implied an existence of already highly developed knowledge from far earlier times (Hartwell, J. L., et al., Antineoplastic Principles in Plants: Recent Developments in the Field, Advances in Pharmacology (1969) 1: 117-209). A fast growing body of evidence obtained in the recent years by utilization of modern scientific, experimental and clinical methods confirms the biological activity of many micro components of plants that can be utilized in prevention or treatment of a variety of chronic diseases, including cancer and cardiovascular disease (Rao, A. V., et al., Anticarcinogenic Effects of Saponins and Phytosterols, American Chemical Society Symposium Series (1997) 662: 313-324; Ghai, G., et al, U.S. Pat. No. 5,955,269; Hartwell, J. L., Types of Anticancer Agents Isolated from Plants, Cancer Treatment Reports (1979) 60(8): 1031-1067; Hutabarat, L. S., et al., Development and Validation of an Isocratic High-performance Liquid Chromatographic Method for Quantitative Determination of Phytoestrogens in Soya Bean, Journal of Chromatography A (1998) 795: 377-382).
Recently there has been visible a prominent trend to replace the conventional medicine approach, heavily dependent on the application of surgical intervention and use of potent synthetic drugs with many detrimental side effects (and thus being perceived by the public as inadequate or even harmful), by using herbal remedies or other forms of nutraceutical supplementation. Many herbal products available on the market are advertised as cures or preventative agents for a wide range of ailments. While a number of these claims might be true, based on their traditional use in folk medicine, for many of them there is little scientific basis underlying the claims of their health benefits.
Despite the fact that scientific evaluation of medicinal plants historically has been responsible for discovery of a multitude of modern medicine, approximately only 1% of plants has been analyzed so far.
When working with medicinal plants, the main goal is to isolate and identify the bioactive constituents. The typical strategy consists of the activity-guided fractionation of the plant extracts, leading to the isolation and identification of the active components. This approach is highly limited, time-consuming and may lead to easily missing any interesting lead compounds that don""t poses the tested activity (Wolfender, J., et al., Comparison of Liquid Chromatography/Electrospray, Atmospheric Pressure Chemical Ionization, Thermospray and Continuous-flow Fast Atom Bombardment Mass Spectrometry for the Determination of Secondary Metabolites in Crude Plant Extracts, Journal of Mass Spectrometry and Rapid Communications in Mass Spectrometry (1995) (Special Issue): S35-S46).
The growing demand of the aging population for high quality herbal supplements offering scientifically confirmed health benefits and presented in a standardized form of known potency, purity and efficacy has created a conducive environment for facilitation of the basic research on the identification of new herbal medicines as well as requirement for development of time and cost effective, reliable quality control analytical testing procedures. The concern of healthcare government agencies for safe and efficacious herbal supplements has led to introduction of much more stringent legislation regulating the allowed medical claims and demanding from the industry presentation of analytical data proving the products purity, potency and efficacy. Thus both, the search for novel, scientifically evaluated herbal medicines and the screening, standardization and quality control of already known herbal remedies, either in the plant material or in different formulations (including extracts, tinctures, suspensions, capsules and compressed tablets) call for a development of a rapid and reliable analytical method.
The analysis of botanical material is not a trivial matter. Usually, a sample to be analyzed contains a very complex mixture of many components. Only some of them might be biologically active, while other may be toxic. Components of these complex mixtures are usually interacting amongst themselves and often work synergistically. Frequently, in many plants, dozens of species and strains of the same genus, differ substantially in content of the active ingredients. Even within the same plant, different parts often have different chemical composition. Furthermore, the presence and concentration of some substances depend greatly on the soil, location, season, time of harvest, storage conditions, handling methods, conditions and solvents used for extraction, etc. This diversity of important conditions affecting the quality of botanical remedies requires therefore implementation of stringent, well-designed and closely-monitored standard operating procedures of manufacturing to ensure consistency from batch to batch of a nutraceutical product, followed by application of an appropriate analysis to ensure consistent potency and efficacy.
The major aims of qualitative analyses in phytochemistry include monitoring of the preparative isolation and purification of phytochemicals, chemotaxonomic testing and drug identification and/or detection of adulterants (Maillard, M. P., et al., Use of Liquid Chromatography-Thermospray Mass Spectrometry in Phytochemical Analysis of Crude Plant Extracts, Journal of Chromatography (1993) 647: 147-154; Games, D. E., Combined High Performance Liquid Chromatography Mass Spectrometry, Biomedical Mass Spectrometry (1981) 8(9): 454-462).
Plant constituents often exist in the form of glycosides. These conjugates may or may not occur together with their respective aglycones. Many glycosides play an important role as drugs and dyes. Glycosides are thermally labile, polar and non-volatile compounds frequently differing in their solubility and biological activity from their respective aglycones. By changing a type and/or number of attached saccharides the physicochemical and biological properties of the glycosides can be modified (Vaccaro, W. D., et al., Sugar-Substituted 2-Azetidinone Cholesterol Absorption Inhibitors: Enhanced Potency by Modification of the Sugar, Bioorganic and Medicinal Chemistry Letters (1998) 8: 313-318). Among phytochemicals existing in the glycosilated form that deserve a special attention due to their wide distribution in nature and a high number of beneficial biological and medicinal properties, are saponins and flavonoid glycosides.
Saponins are glycosides that commonly occur in higher plants where they are generally found in the roots, flowers and seeds. They are biosynthesized by more than 500 species belonging to almost 100 different families (Price, K. R., et al., The Chemistry and Biological Significance of Saponins in Foods and Feedingstuffs, Critical Reviews in Food Science and Nutrition (1987) 26(1): 27-135). They are also found in many marine organisms. Saponins belong to one of two groups depending on the structure of their aglycone moiety (sapogenin): the triterpine group, in which the aglycone is usually represented by oleanane, ursane or damarane skeleton, and the steroid group. The latter also includes the steroid alkaloids. The most common sugars encountered in saponins are hexoses (glucose, galactose and mannose), 6-deoxyhexoses (rhamnose), pentose (arabinose and xylose), uronic acids (glucuronic acid and galacturonic acid) or amino sugars (glucosamine and galactosamine) (Fang, S., et al., Rapid Analysis of Steroidal Saponin Mixture Using Electrospray Ionization Mass Spectrometry Combined with Sequential Tandem Mass Spectrometry, Rapid Communications In Mass Spectrometry (1998) 12: 589-594). Sugars may be linked to the sapogenin at one or two glycosylation sites (through an ether or/and an ester linkage), giving the corresponding monodesmodic or bidesmosidic saponins, respectively (Maillard, M. P., et al., Determination of Saponins in Crude Plant Extracts by Liquid Chromatography-Thermospray Mass Spectrometry, Journal of Chromatography (1993) 647: 137-146; Lee, M., et al., Analysis of Saponins from Black Bean by Electrospray Ionization and Fast Atom Bombardment Tandem Mass Spectrometry, Journal of Mass Spectrometry (1999) 34: 804-812).
Because of the glycosylation of their hydrophobic aglycones, saponins act as biological detergents and, when agitated with water, form a soapy lather that gives rise to name of this group of compounds. From a biological point of view saponins have diverse group properties, some deleterious, but many beneficial (Van Setten, D. C., et al., Multiple-Stage Tandem Mass Spectrometry for Structural Characterization of Saponins, Analytical Chemistry (1998) 70(20): 4401-4409). Some saponins have been used as plant drugs in folk medicine. They may exhibit cardiac activity, hemolytic activity, activity as fish poisons, hypocholesterolemic (Deninno, M. P., U.S. Pat. No. 5,698,526), immunostimulatory and anti-tumorigenic activity (Hostettmann, K., et al., Saponinsxe2x80x94Chemistry and Pharmacology of Natural Product, Cambridge University, Cambridge (1995); Fuzzati, N., et al., Identification of Soyasaponins by Liquid Chromatographyxe2x80x94Thermospray Mass Spectrometry, Journal of Chromatography A (1997)777: 233-238); Lee, M., et al., Analysis of Saponins from Black Bean by Electrospray Ionization and Fast Atom Bombardment Tandem Mass Spectrometry, Journal of Mass Spectrometry (1999) 34: 804-812). They can be used as bitterness and sweetness modifiers, allelochemicals and cosmetic ingredients. Saponins have a potential as pharmaceutical synthons (Mostad, H. B., et al., Separation and Characterization of Oleanene-type Pentacyclic Triterpenes from Gypsophila Arrostii by Liquid Chromatography-Mass Spectrometry, Journal of Chromatography (1987) 396: 157-168) and have been used in hormone synthesis (Hardman, R., Board of Pharmaceutical Sciences (editor), Conception and Contraception, Exerpta Medica, Amsterdam, (1975) p. 60).
The second important class of phytochemicals which attracted a high interest due to its wide distribution in nature and diversified biological properties are flavonoids. These polyphenolic compounds, apart from catechins and proanthocyanidins, consist mainly of glycosides of flavonols, flavons, flavanones, anthocyanins and less frequently isoflavons or free aglycones. Flavonoids play important role in the ecology of plants. Because of their attractive colors, flavonols, flavons, and anthocyanidins are likely to be a visual signal for pollinating insects. Catechins and other flavonols have astringent properties and they act as feeding repellants, while isoflavones are important plant-protective phytoalexins (Pietta, P., Flavonoids in Medicinal Plants, Antioxidant Health Dissertation (1998), 7 (Flavonoids in Health and Disease), 61-100). Flavonoids represent an important constituent of many edible plants and are present in foods and beverages derived from plants.
Some flavonoid-containing species have been used in traditional medicine. Recently these phytomedicines have been extensively investigated and their health benefits confirmed in many cases for the long-term treatment of mild and chronic diseases or in attaining and maintaining a condition of well-being, (Pietta, P. G, et al., Fitomedicine e Nutrienti, Verona: Ricchiuto G M, (1996)). Flavonoids function as strong antioxidants (Bors, W., et al., Radical Chemistry of Flavonoid Antioxidants, Antioxidants in Therapy and Preventative Medicine (1990) 264: 165-170; Budzianowski, J., et al., Studies on Antioxidative Activity of Some C-Glycosylflavones, Polish Journal of Pharmacology and Pharmacy (1991) 43: 395-401), free-radical scavengers (Lonchampt, M. et al., Protective Effect of a Purified Flavonoid Fraction against Reactive Oxygen Radicals, Arzneimittelforschung (1989) 39(8): 882-885), and metal chelators and their biological properties can also be linked with their interaction with enzymes, adenosine receptors, and biomembranes (Saija, A., et al., Flavonoids as Antioxidant Agents: Importance of their Interaction with Biomembranes, Free Radical Biology and Medicine (1995) 19(4): 481-486). Many of the bioflavonoids exhibit very beneficial pharmacological activities, such as anti-inflammatory, antiallergic, antimicrobial, antioxidative, enzyme-inhibitory effects, etc. (Von Wacker, A., Antivirale Wirkung von Pflanzeninhaltsstoffen, Arzneimittelforschung (1978) 28(3): 347-350; Frazier, S. E., U.S. Pat. No. 4,238,483; Havstee, B., Biochemical Pharmacology (1983) 32: 1141; Pathak,D., et al., Fitoterapia (1991) 62:371).
The identification of individual flavonoids, sapogenins and their glycosides has long been carried out by Mass Spectrometry (Dawidar, A. M., et al., Mass Spectra of Steroid Saponins, Journal of Pharmaceutical Sciences (1974) 63: 140-142; Harborn and Williams. The Flavonoids. Ed, Harborn, J. B. et al., Chapman and Hall, (1975): 376-441; Franski, R., et al., Application of Mass Spectrometry to Structural Identification of Flavonoid Monoglycosides Isolated from Shoot of Lupin (Lupinus Luteus L.), Acta Biochimica Polonica (1999) 46(2): 459-478) and Ultraviolet Spectroscopy (Nakaori, T., et al, Journal of Pharmaceutical Sciences Japan. (1956) 76: 323; Lunte, S. M., Structural Classification of Flavonoids in Beverages by Liquid Chromatography With Ultraviolet-visible and Electrochemical Detection, Journal of Chromatography (1987) 384: 371-382; Tsuchiya, H., High-Performance Liquid Chromatographic Analysis of Polyhydroxyflavones using Solid-Phase Borate-Complex Extraction, Journal of Chromatography B (1998) 720: 225-230). More recently 13C-NMR has been used for the structural elucidation of flavonoid glycosides Guinaudeau, H., et al., Phytochemistry (1981) 20: 1113; Hosny, M., Novel Isoflavone, Cinnamic Acid, and Triterpenoid Glycosides in Soybean Molasses, Journal of Natural Products (1999) 62(6): 853-858). These techniques were executed on highly purified compounds and were not applied to mixtures.
Separation of individual flavonoids, sapogenins and their glycosides from each other has long been carried out by Paper, Thin Layer and Open Column Chromatography (Harborn, J. B., et al, Flavone and Flavonol Glycosides. Flavonoids: Advances in Research (1982) 261-311. Editor: Harborne, J., Wolf, W. J., et al, Journal of American Oil Chemists Society. (1970) 47: 89).
More recently HPLC has been used for the separation of individual flavonoids, sapogenins and their glycosides from each other (Stewart et al., Biochemical Systems Ecology, (1980) 8: 119; Galensa, R., et al, Analyse von Flavonoidglycosiden durch Hochdruck-Flusskeits-Chromatographie, (1978) 166: 355-358; Domon, B., Journal of Chromatography (1984) 315: 441). These techniques gave limited resolution between individual glycosides of either the flavonoids or the sapogenins. Selectivity between the flavonoid and sapogenin classes of compounds was effected through chemical purification steps or separate preparative chromatographic steps.
The combination of HPLC and Diode Array UV-Visible Detection gave new possibilities in qualitative analysis of flavonoids in plant extracts (Hasler, A., et al., High-performance Liquid Chromatographic Determination of Five Widespread Flavonoid Aglycones, Journal of Chromatography (1990) 508: 236-240; Inada, S., et al., U.S. Pat. No. 4,968,787 and 39th Annual Congress on Medicinal Plant Research September 1991). Information about the type and number of glycosidic units was lost due to the preparation. The extraction and purification did not address the identification and quantization of individual glycosides. Mass Spectrometry with Thermospray Ionization permitted routine online analysis of a number of glycosides of both flavonoid and sapogenin classes, but sensitivity was limited and interpretation was complicated by the frequent formation of artifacts (Iida, J., et al., Application of Thermospray Liquid Chromatography/Mass Spectrometry to Analysis of Glycosides, Analytical Sciences (1991) (Supplement): 963-966; Maillard, M. P., et al., Thermospray LC-MS Analysis of Saponins in Crude Plant Extracts, Planta Medica (1992) 58(Supplement Issue 1): A 673; Wolfender, J. L., et al., Liquid Chromatographic-Thermospray Mass Spectrometric Analysis of Crude Plant Extracts Containing Phenolic and Terpene Glycosides, Journal of Chromatography (1993) 647: 183-190; Wolfender, J., et al., Comparison of Liquid Chromatography/Electrospray, Atmospheric Pressure Chemical Ionization, Thermospray and Continuous-flow Fast Atom Bombardment Mass Spectrometry for the Determination of Secondary Metabolites in Crude Plant Extracts, Journal of Mass Spectrometry and Rapid Communications in Mass Spectrometry (1995) (Special Issue): S35-S46; Pietta, P., et al., Thermospray Liquid Chromatography-Mass Spectrometry of Flavonol Glycosides From Medicinal Plants, Journal of Chromatography A (1994) 661: 121-126). Moreover, due to the relatively energetic ionization of the thermospray technique the higher glycosides were not observed. Few such compounds above 850 Da were reported with this technique. Continuous Flow Fast Atom Bombardment gave some advantages in ionization of small polar molecules but at the cost of instrumental complexity and reliability (Wolfender, J., et al., Comparison of Liquid Chromatography/Electrospray, Atmospheric Pressure Chemical Ionization, Thermospray and Continuous-flow Fast Atom Bombardment Mass Spectrometry for the Determination of Secondary Metabolites in Crude Plant Extracts, Journal of Mass Spectrometry and Rapid Communications in Mass Spectrometry (1995) (Special Issue): S35-S46; Wolfender, J. L., et al., Liquid Chromatography Combined with Thermospray and Continuous-flow Fast Atom Bombardment Mass Spectrometry of Glycosides in Crude Plant Extracts, Journal of Chromatography A (1995) 712: 155-168).
The advent of Electrospray Ionization permitted molecules to be ionized with very low energies under atmospheric pressures and at room temperatures. Very polar, high molecular weight species could be routinely analyzed with little artifact formation that could complicate interpretation (Hakkinen, S., et al., High-performance Liquid Chromatography with Electrospray Ionization Mass Spectrometry and Diode Array Ultraviolet Detection in the Identification of Flavonol Aglycones and Glycosides in Berries, Journal of Chromatography A (1998) 829: 91-100; Schopke, T., et al., Application of MS-MS for the Rapid, Comparative Analysis of Saponin Mixtures as Exemplified by the Deacylated and Partially Deacylated Triterpenoid Saponins of Bellis Annua, Planta Medica (1996) 62: 336-340; Mauri, P. L., et al., Liquid Chromatography/Electrospray Ionization Mass Spectrometric Characterization of Flavonol Glycosides in Tomato Extracts and Human Plasma, Rapid Communications in Mass Spectrometry (1999) 13: 924-931; Stobiecki, M., et al., Detection of Isoflavonoids and their Glycosides by Liquid Chromatography/Electrospray Ionization Mass Spectrometry in Root Extracts of Lupin (Lupinus Albus), Pytochemical Analysis (1999) 10: 198-207). The technique also permits the use of Collision Induced Fragmentation for generating ions that aids in structure elucidation. Gelpi, E., Biomedical and Biochemical Applications of Liquid Chromatography-Mass Spectrometry, Journal of Chromatography A (1995) 703:59-80. Negative ion mode in Electrospray Ionization was also shown to have advantages (Wolfender, J., et al., Comparison of Liquid Chromatography/Electrospray, Atmospheric Pressure Chemical Ionization, Thermospray and Continuous-flow Fast Atom Bombardment Mass Spectrometry for the Determination of Secondary Metabolites in Crude Plant Extracts, Journal of Mass Spectrometry and Rapid Communications in Mass Spectrometry (1995) (Special Issue): S35-S46; Watson, D. G., et al., Analysis of Flavonoids in Tablets and Urine by Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Mass Spectrometry, Rapid Communications in Mass Spectrometry (1998) 12: 153-156).
The combination of three powerful techniques LC/DAD/ESIMS was used to study the aglycones and glycosides present in berries (Hakkinen, S., et al., High-performance Liquid Chromatography With Electrospray Ionization Mass Spectrometry and Diode Array Ultraviolet Detection in the Identification of Flavonol Aglycones and Glycosides in Berries, Journal of Chromatography A (1998) 829: 91-100; Justesen, U., et al., Quantitative Analysis of Flavonols. Flavones, and Flavanones in Fruits, Vegetables and Beverages by High-performance Liquid Chromatography with Photo-diode array and Mass Spectrometric Detection, Journal of Chromatography (1998) 799: 101-110). These works however largely concentrated on the identification of the flavonoid aglycones or of glycosides of no greater than two units.
The present invention provides a fast and reliable method for the simultaneous analysis of both flavonoid glycosides and steroidal glycosides in one procedure. As a model to show the usefulness of this technique we have chosen plants from Hosta genus which belongs to the subfamily Asphodeloideae in Liliaceae. These plants are widely distributed thus offering easy and economical access to this source of flavonoid and steroidal glycosides of potential medicinal application. The young leaves and buds of the plants are edible and the leaves and rhizomata have been used as a folk medicine in China and Japan (Jiang Su New Medical College (ed.), xe2x80x9cDictionary of Traditional Chinese Crude Drugsxe2x80x9d, vol.1, Shanghai Scientific Technologic Publishers, Shanghai, (1977), p.557). A steroidal saponin identified as hexasaccharide and prepared from the extract of dried Hosta leaves by O. Masamitsu, et al. (Ochi, M., et al., Steroid Saponin from Hosta and Antimicrobial and Antitumor Agents Containing It, JP 10 114,791 [98 114,791] (C1. C07J71/00), May 6, 1998, Appl. 96/270,292, Oct. 11, 1996; 12 pp; CA 129: 32293w; Ochi, M., et al., Novel Steroidal Saponin and Antimicrobial Agents and Antitumor Agents Containing It, JP 10 158,295 [98 158,295] (C1. C07J71), Jun. 16, 1998, Appl. 96/320,142, Nov. 29, 1996; 12 pp; CA 129:113511t) exhibit antibacterial and antitumor activity while some of the steroidal glycosides identified by M. Mimaki group displayed cytostatic activity on HL-60 cells.
Although eight kaempferol glycosides (Budzianowski, J., Kaempferol Glycosides from Hosta Ventricosa, Phytochemistry (1990) 29(1): 3463-3467) and twenty six steroidal glycosides (Takeda, K., et al., Studies on the Steroidal Components of Domestic Plantsxe2x80x94XLVI Constituents of Hosta Species (3) xcex9425(27)-Sapogenins, Tetrahedron, (1965) 21: 2089-2093; Takeda, K., et al., Studies on Biochemical Transformation of Plant Steroids, Part Biochemical Interconversion of the xcex9425(27)-and the Saturated 25D- or 25L-Sapogenins, Journal of Chemical Society C (1967) (9): 876-882; Takeda, K., et al., Studies on Biochemical Transformation of Plant Steroids, Part II. Biochemical Conversion of Gitogen into 12-Oxygenated Sapogenins in Hosta Kiyosumiensis, Chemical and Pharmaceutical Bulletin (1968) 16(2): 275-279; Mimaki, Y., et al., Steroidal Saponins from the Underground Parts of Hosta Longpipes and Their Inhibitory Activity on Tumor Promoter-Induced Phospholipid Metabolism, Chemical and Pharmaceutical Bulletin (1995) 43(7): 1190-1196; Mimaki, Y., et al., Steroidal Saponins from Hosta Longpipes and Their Inhibitory Activity on Tumor Promoter-Induced Phospholipid Metabolism of HeLa Cells, Phytochemistry (1996) 42(4): 1065-1070; Mimaki, Y., et al., Steroidal Glycosides from the Underground Parts of Hosta Plantaginea Var. Japonica and Their Cytostatic Activity on Leukemia HL-60 Cells, Phytochemistry (1997) 44(2): 305-310; Ochi, M., et al., Steroid Saponin from Hosta and Antimicrobial and Antitumor Agents Containing It, JP 10 114,791 [98 114,791] (C1. C07J71/00), May 6, 1998, Appl. 96/270,292, Oct. 11, 1996; 12 pp; CA 129: 32293w; Mimaki, Y., et al., Steroidal Saponins from the Rhizomes of Hosta Sieboldii and Their Cytostatic Activity On HL-60 Cells, Phytochemistry (1998) 48(8): 1361-1369) have been previously separated from Hosta leaves and Hosta rhizomers, respectively, there has been no reports of any comprehensive procedure to extract simultaneously both classes of glycosides from the Hosta leaves.
The eight reported kaempferol glycosides after separation were analyzed and identified individually by TLC, and UV, 1H and 13C NMR spectroscopies.
Similarly, the previously reported steroidal glycosides extracted from Hosta root after separation and purification were analyzed and identified individually by NMR, IR and in some cases by negative-ion FAB-MS spectrometry.
The present invention provides a method for the simultaneous analysis of both flavonoid glycosides and steroidal glycosides in one procedure. Compounds glycosidic units from 0 to 4 or more can be readily identified for both compound classes. In the preferred embodiment the method is comprised of an extraction and a pre-purification step followed by an instrumental technique (LC/DAD/ESI/MS) that allows for accurate qualitative and quantitative measurements. In another embodiment the pre-purification step is eliminated from the procedure. The resulting method is useful for screening functions and gives detailed data in the shortest amount of time.